The fluorescence detection of superoxide radical using hydroethidine could be complicated by the presence of heme proteins.

This study shows that hydroethidine (HE) used for the qualitative detection of superoxide anion can also be oxidized by heme proteins such as the mitochondrial cytochromes, hemoglobin, and myoglobin, forming spectrally nonhomogenous mixtures of HE-derived products of various oxidation states. All oxidation products show excitation/emission peaks (490-495/580-600 nm) near the excitation/emission peaks (475/570 nm) of the HE-superoxide oxidation product, and this may pose serious interference problems to the fluorescent detection of the superoxide radical. This paper discusses possible precautionary steps that should be taken to minimize the interfering problems in the HE-superoxide assay and suggests its use mainly for reactive oxygen species detection.